A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

Abstract

Cell-free transcription-translation systems are a versatile tool to study gene expression, enzymatic reactions and biochemical regulation mechanisms. Because cell-free transcription-translation systems are often derived from cell lysates, many different substances, among them amino acids, are present. However, experiments concerning the incorporation of non-canonical amino acids into proteins require a system with negligible amounts of canonical analogs. Here we propose a two-step method for the removal of residual free lysine in an all Escherichia coli-based cell-free expression system. The first step consists of the expression of a high-lysine dummy protein. The second step consists of direct removal via binding between lysine and DNA. The presented method is an efficient, fast and simple way to remove residual lysine without altering the system ability to perform gene expression.